Evaluation of antimicrobial, antiquorum sensing, and cytotoxic activities of new vanillin 1,2,3-triazole derivatives

Abstract Vanillin (1), the main constituent of vanilla species, was used as a starting natural scaffold for the synthesis of five new (2–6) and one known (7) triazole derivatives via click chemistry using the copper (I)-catalyzed azide–alkyne cycloaddition method. Vanillin and its new derivatives; 4-{1-[2-Hydroxymethyl-5-(5 methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-tetrahydro-furan-3-yl]-1H[1,2,3]triazol-4-ylmethoxy}-3-methoxy-benzaldehyde (2); [4-(4-Formyl-2methoxy-phenoxymethyl)-[1,2,3]triazol-1-yl]-acetic acid methyl ester (3); 4-[1-(4-Acetyl-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-3-methoxy-benzaldehyde (4); 4-[4-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethoxy)-3-methoxy-phenyl]-but-3-en-2-one (5); and 4-[4-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethoxy)-3-methoxy-phenyl]-4-hydroxy-butan-2-one (6), as well as the previously known derivative (7) were subjected to antimicrobial, antiquorum-sensing and cytotoxic evaluation. Compounds 4–7 possessed the most notable enhancement in the anti-bacterial activity against Bacillus cereus, Pseudomonas aeruginosa and antifungal activity against Candida albicans. However, compounds 1 and 2 exhibited high antiquorum-sensing activity against Chromobacterium violaceum using catechin as a positive control. Compounds 4–7 demonstrated selective cytotoxicity against MCF-7 and HepG2 cancer cells compared to normal lung fibroblast cells (WI-38). These findings proved the usefulness of synthesis bioactive derivatives from vanillin through chemical modifications. Graphical Abstract


Introduction
Natural products have a wide range of diversity, high incomparable chemical diversity and novel mechanisms of action, and they have continued to play a pivotal role in many drug development and research programs (Hong 2011). An ample interest has been possessed in the modification of available natural products; either chemically or biologically in an attempt to obtain new analogues with different and/or enhanced activities. Many synthetically modified natural products have been successfully developed for clinical application to treat human diseases (Baker et al. 2007;Cragg and Newman 2013).
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most abundant aroma ingredients of the vanilla species. It has been extensively used for the preparation of cakes, soft drinks, ice creams, chocolates, liquors, perfumes, pharmaceuticals and nutraceuticals (Ranadive 1992;Zamzuri and Abd-Aziz 2013). In the recent past, it has been subjected to rigorous research and it has proven its potential to be a pharmacotherapeutic agent in several diseases like depression, Alzheimer's and Parkinson's diseases (Anand et al. 2019). It has been reported to exhibit antioxidant, antitumor, antimicrobial and antimelanogenesis activities (Fitzgerald et al. 2004;Pedroso et al. 2013;Raut et al. 2013;Ashraf et al. 2015).
Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) process has developed as the premier example of click chemistry reactions, which described as a set of perfect bond-forming reactions useful for easy, rapid assembly of molecules with desired function) Kolb et al. 2001;Liang and Astruc 2011). It acts as the most efficacious chemical ligation tool for building bioactive compound libraries because of its superior stereo-selectivity and capacity to give pure products for drug screening (He et al. 2012). As reaction products of CuAAC, 1,2,3-triazoles are attractive constructs of target molecules due to their wide range of biological activities, such as antimicrobial, antitubercular, antimalarial, anticancer, and antioxidant (Tiwari et al. 2016). The reported triazole-based compounds revealed low toxicity and multi-drug resistances with high bioavailability, good pharmacokinetics property, diversity of drug administration and better curative effect (Zhou and Wang 2012). Recent researches focussed on the chemical modifications of functional groups of some natural products like thymol, Curcumin to produce new 1,2,3-triazole hybrids with enhanced pharmacological effects (Seghetti et al. 2020;Alam et al. 2021;Almalki et al. 2021).
Based on these considerations, we designed and synthesised new 1,2,3-triazole based molecules from commercially available natural starting material (vanillin) with a high overall yield. Furthermore, the antimicrobial, antiquorum-sensing and cytotoxic activities of vanillin derivatives were determined and compared to vanillin activities.

Chemistry and compounds identification
The structure of vanillin (1) was identified by comparing its IR, 1 H-NMR and APT spectral data (Figures S1-S3 and Table S1) with that reported in the literature (Kale et al. 2013). Vanillin was converted to mono-alkyne vanillin by using propargyl bromide according to Batool et al. (2014) (Supplemental material file S1.4.1.). Mono-alkyne vanillin was obtained as white needle crystals with m.p. 81.0-82.5 C and R f ¼ 0.41 (petroleum ether-ethyl acetate 8:2, v/v). Its IR (KBr) spectrum ( Figure S4) exhibited characteristic peaks at t max 3247 and 2123 cm À1 revealed the presence of a terminal alkyne. The suppression of OH stretching peak respect to vanillin was confirming alkylation of the phenolic OH group of vanillin with propargyl bromide and this support the formation of mono-alkyne vanillin (Sun and Wu 2010). The latter was separately reacted with zidovudine, methyl 2-azidoacetate, 4-azido acetophenone, and benzyl azide to produce compounds 2, 3, 4 and (5-7), respectively ( Figure 1). Careful study of IR, 1 H-NMR and APT spectral data of these compounds confirmed the successful formation of a triazole ring in each compound. IR spectral data (KBr) (Figures S5-S9) of compounds 2-6 showed a characteristic peaks at t max ranging from 3146 to 3140 and from 1588 to 1595 cm À1 suggesting the presence of triazole ring in each compound (Silverstein et al. 1991). 1 H-NMR data of compounds 2-6 (Figures S10-S14) showed the presence of methine proton singlets (H-5 0 ) at d H ranging from 8.46 to 7.56 and methylene proton singlets (H-8) at d H ranging from 5.51 to 5.27 indicating the formation of triazole ring and the presence of methylene bridge between this ring and vanillin moiety. This was confirmed by their APT and 13 C-NMR spectral data of compounds 2-6 (Figures S15-S19) which revealed the presence of methine carbon signals (C-5 0 ) at d C ranging from 121.7 to 124.8 and a quaternary carbon signal (C-4 0 ) at d C ranging from 142.2 to 144.4 of triazole ring in addition to downfield methylene carbon signal (C-8) at d C ranging from 61.7 to 63.1 (Anand and Kulkarni 2017). The structure of compound 2 was also confirmed by its ESI-MS data ( Figure S20) and by comparing its NMR spectral data (Figures S10 and S15) with the previous literatures of its parent's compounds vanillin (Kale et al. 2013) and zidovudine (Sethi 1991; Figures S1-S3 & S21-S22 and Tables S1 & S2). The 1 H-NMR and APT spectral data of compound 3 (Figures S11 and S16) revealed the presence of acetic acid methyl ester moiety which established from the downfield shifted carbonyl signal at d C 166.5 (C-1 00 ), methoxy group at d H/C 3.80/53.1 and methylene group at d H/C 5.17/50.7 ( Figures S20-S22). The final structure of compound 3 was confirmed from its ESI-MS data ( Figure S23). The presence of acetophenone moiety in compound 4 was deduced from the APT spectral data ( Figure S17) which exhibited upfield methyl carbon signal at d C 26.6 (CH 3 , C-8 00 ) and downfield carbonyl group signal at d C 196.4 (C-7 00 ) in addition to carbon signals at d C 120.0 (C-2 00 /6 00 ), 129.9 (C-3 00 /5 00 ), 136.9 (C-1 00 ) and 139.7 (C-4 00 ). This was confirmed by the presence of methyl proton singlet at d H 2.65 (H-8 00 ) and two ortho coupled proton signals each integrated for two protons at d H 7.87(d, J ¼ 9 Hz, H-2 00 & H-6 00 ) and 8.12 (d, J ¼ 9 Hz, H-3 00 & H-5 00 ). The structure of 4 was also confirmed from its ESI-MS data ( Figure S24).

Antimicrobial activity
The in-vitro antibacterial activity of vanillin (1) and its derivatives (2-7) was evaluated against Gram-positive bacteria (B. cereus and S. aureus), Gram-negative bacteria (E. coli and P. aeruginosa) and a pathogenic fungus C. albicans. The minimum inhibitory concentration (MIC) of compounds 1-7 (Table S4) was determined against tested bacterial isolates as reported by Clinical Laboratory Standard Institute (CLSI) (2015) and against tested fungal isolate as reported by CLSI (2008). Results indicated that vanillin derivatives (5-7) showed potent activity against B. cereus with MICs range from 50 to 200 mg/mL, while derivatives 1-4 exhibited moderate to week effect against B. cereus. All compounds showed low antibacterial activity against S. aureus and E. coli with MICs in range of >1600-400 mg/mL when compared with Ampicillin/Clavulanic acid results (Table S4). Derivatives (4-7) showed strong activity against P. aeruginosa with MICs of 200-400 mg/mL compared to Ampicillin/Clavulanic acid (MIC; 512 mg/mL). However, the parent compound (1) and derivatives (2-3) had the lowest activity (MIC; 1600 mg/mL). The antifungal activity results revealed that vanillin (1) had antifungal activity against C. albicans in accordance with the results of Raut et al. (2013). Compound 1 and its derivatives (5-7) showed the highest antifungal activity among the tested compounds with MIC 200 mg/mL compared to standard Fluconazole (MIC; 400 mg/mL) against the tested isolate. Furthermore, compounds 2 and 3 exhibited reasonable antifungal activity with MIC of 800 mg/mL, while compound 4 had weak activity against C. albicans (Nunes et al. 2016).

Antiquorum-sensing activity
The antipathogenic potential screening was checked by examining the antiquorumsensing activity of the tested compounds (1-7) against C. violaceum using catechin as a positive control and DMSO as a negative control. The antiquorum-sensing activity was determined by testing their ability for inhibiting the release of a violet pigment (violacein), which is released in response to acyl homoserine lactones signals (McLean et al. 2004;Chu et al. 2011). Results (Table S4), obtained by measuring the pigment inhibition radius revealed that the vanillin and its derivative 2, showed the highest antiquorum-sensing activity, while the other derivatives showed no activity. Finally, the synergistic effect in antimicrobial activity was observed when vanillin and 1,2,3-triazole are conjugated. Triazole derivatives 4-7 containing N-substituted benzene ring possessed the most notable enhancement in the anti-bacterial activity against Bacillus cereus, Pseudomonas aeruginosa and antifungal activity against Candida albicans this is in agreement with previous literatures (Lal et al. 2018).

General details
See supplemental material file.

Antimicrobial, antiquorum sensing and MTT cytotoxic assays
See supplemental material file.

Conclusion
Inspired by the click chemistry and prompted by the chemotherapeutic importance of 1,2,3-triazoles as antimicrobial and cytotoxic agents, we have synthesised five new and one known vanillin-triazole derivatives and evaluated for their antimicrobial, antiquorum-sensing and cytotoxic activities. Compounds 5-7 showed enhanced antimicrobial activity than vanillin (1) with good selectivity value (IC 50 /MIC > 10) using normal lung cell line WI-38. Moreover, compound 2 showed higher antiquorum-sensing activity with respect to Catechin. Compounds 4-7 showed the highest cytotoxic activity against two cancer cell lines MCF-7 and HepG2 with high selectivity indices (2.66-4.99). Finally, vanillin 1,2,3-triazole derivatives reported herein may be useful in managing such microbial infections in addition to breast and hepatic carcinoma.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.